1. Field of the Invention
The present invention relates to measurements of contaminants in the soil and other organic or environmental materials, using a biologically relevant chemical analysis that will measure the amount of contaminants in a given sample that may be expected to be absorbed by a human being ingesting the contaminated soil.
2. Description of the Related Art
The clean up of hazardous wastes in the environment has become a scientific and socially significant problem. As more effort is spent on the clean-up of contaminated environmental sites and other areas of pollution, the need to monitor the efficiency and efficacy of these clean-up methods becomes more pressing.
In many instances, the technical ability to detect contaminating compounds exceeds our ability to remove them. For example, in soil that has been contaminated with petroleum products, these products and their breakdown products can be detected to very minute levels using analytical chemical techniques such as mass spectrometry and gas chromatography (GC/MS). It is not currently possible to remove these breakdown products to an undetectable level. Breakdown products known as polycyclic aromatic hydrocarbons (PAH's) are major concerns in petroleum contamination, due to their carcinogenic effect. This effect is mediated by the oral ingestion of materials, such as soil, which contain small amounts of the PAH's. Children are particularly vulnerable to soil ingestion.
PAH's may consist of 2- to 6-ring polycyclic compounds; their composition varies with the type of contaminated soil.
The standard prior art technique for measuring these contaminants in soil is to extract a soils sample with methylene chloride or other organic solvents or use sonication; separate aliphatics and aromatics according to cleanup methods, and subject all fractions to GC/MS analysis under EPA guidelines.
This regulatory-based approach simplifies risk and exposure assessment by assuming that all contaminants in ingested soil are completely absorbed into the blood stream, i.e. are 100% bioavailable.
This simplified assumption has been used conservatively to assure the protection of public health, particularly for those who have been involuntarily exposed to the contaminants. However, especially for sites that were contaminated years ago, the assumption of 100% bioavailability could overestimate the ecological risk and increase remediation expenses substantially with negligible reduction of risk. Field and laboratory studies have demonstrated that the bioavailability of contaminants from soil to microorganisms and small animals can be significantly less than 100%.
There are three known in vitro approaches for estimating the bioavailability of environmental contaminants in the GI tract: (1) the two stage physiologically based extraction procedure; (2) the everted sac technique and (3) brush border membrane vesicles.
The two-stage physiologically-based extraction procedure employs an extraction procedure designed to simulate the stomach and small intestinal stages of digestion and adsorption of metal ions. The method generally uses solutions of specific pH that contain digestive enzymes (e.g. pepsin in the stomach, pancreatic enzymes and bile acids in the small intestine) mixed with test substrates (e.g. food and soil) to reproduce GI tract function and chemistry. Depending on the GI emptying conditions, a value of pH 1.35-2.0 is selected for the gastric incubations, and pH 7.0-7.5 is selected for the small intestinal incubations. This in vitro method may be simplified by assuming that enzymes have minor influence on the release of metals from soil matrix. At the end of the incubation period, the sample is removed from the incubation flasks, centrifuged, and the supernatant is analyzed. Recently, a continuous flow in vitro method to estimate the bioavailability of zinc and calcium from foods was developed. This method employs a simulated gastric digestion with pepsin, gradual pH change during the first 30 minutes of dialysis in an Amicon stirred cell, and a further two hour of continuous dialysis accompanied by intestinal digestion with pancreatin-bile extract. The percentage of continuously dialyzed metal ions was used to express the bioavailability.
The second approach is based on an everted sac that is prepared by cutting a small segment of the intestine from laboratory animals, everting the segment, filling the sac with 0.5 to 1.0 ml of oxygenated physiological buffer solution, tying off both ends of the sac and incubating it in a well-oxygenated buffer solution containing the test substrates at 37.degree. C. for 15 to 60 minutes. When necessary, the incubating buffer solution would also contain a nonpermeable marker to correct for the volume adherent to the mucosa. The everted sac technique has been used to study the gastrointestinal absorption of inorganic mercuric compounds, aromatic hydrocarbons, and the transport of copper.
The third method involves the isolation of brush border membrane vesicles from intestinal cells. Unlike the everted gut sac technique, this method involves the disruption of the cellular structure followed by either density gradient centrifugation, free-flow electrophoresis, immunoabsorbent chromatography, or precipitation of nonbrush-border membranes through the addition of Ca.sup.++ or Mg.sup.++. These isolated membrane vesicles have transport properties similar to in vivo conditions. During the bioavailability experiment, membrane vesicles are incubated in buffer containing the test substrate. At the end of the incubation duration, membrane vesicles are filtered, washed thoroughly, and analyzed for the amount of chemicals being retained. This method has been used for in vitro measurement of absorption of inorganic mercury, the transport and absorption of zinc and the uptake of iron in the intestine. The three categories of in vitro methods described above are mostly designed for single application, include a limited number of simulated parameters, and are not directly applicable for simulating the bioavailability of petroleum, (i.e., hydrophobic) hydrocarbons in human GI tract. The first category is designed mainly for measuring the bioavailability of metals whose solubility is mainly controlled by the pH and mixing intensity. The other two categories are mainly for measuring only the transport and adsorption of chemicals through the intestinal membranes.
When measuring bioavailability of complex mixtures of contaminants, it is important to remember that the human GI tract consists of different anatomical regions with very different biophysico-chemical conditions. For example, petroleum products are a mixture of different classes of organic compounds, and each class may behave differently in different anatomical regions of the human GI tract, thereby influencing the absorption, metabolism, and the ultimate bioavailability to humans. Under these dynamic conditions a more versatile in vitro approach is required. Relevant in this regard are in vitro GI models based on techniques developed for nutritional, pharmacology, and pharmacokinetics use.
Known in vitro pharmacological GI models include (1) the computerized gastro-intestinal model (2) the simulated human intestinal microbial ecosystem (SHIME) (3) the "artificial stomach" and improved dialysis cell (IDC) by Saroie and Gauthier, (1968), J. Food Science 51(2): 494-498. The first category is designed for measuring the bioavailability of metals whose solubility is mainly controlled by the pH and mixing intensity. The other two methods are for measuring only the transport and adsoprtion of chemicals through the intestinal membranes.
In the first model, a multi-compartmental computer-controlled in vitro model was designed to simulate the transit time and dynamic physiological processes occurring within the lumen of the gastrointestinal tract of man and monogastric animals. The computer model implements measurements from in vitro studies to prescribe the biophysico-chemical conditions, and uses exponential equations to control the transit time of chyme in the GI tract. The ability of the model for reproducing in vitro data on meal transit, pH, bile salt concentrations and the absorption of glucose was tested.
SHIME is a 5-step multi-chamber reactor designed mainly to simulate the gastro-intestinal microbial ecosystem in human see (Minekos, et al., A.T.L.A. 23:197-209 (1995)). The small intestine is simulated by a two-step "fill and draw system", and the large intestine by a three-step reactor. The medium used in the reactor system is similar to that of the human gastro-intestinal tract. SHIME has been tested by monitoring fermentation fluxes and products. Measurements show that resulting patterns of microbial diversity and activity are similar to those observed from in vitro studies.
The artificial stomach is an in vitro model which stimulates the digestion of milk protein. It involves the use of a reaction vessel inside a shaken water bath. Computer-controlled peristaltic pumps continuously provided, at a variable rate, additional enzymes and HCl to the reactor, and allowed the collection of digested products. The in vitro results showed good relations with in vitro data.
The artificial stomach model may also be constructed to simulate the gastric secretion and emptying in physiological situations specifically for antacid evaluation. It involves the use of a `gastric` reservoir and a peristaltic pump to imitate the interaction between secretory flux and variation in emptying fluxes, the presence of proteins, and the human gastric juice of different pH. Measurements obtained from this model show good agreement with clinical data.
IDC is a dialysis cell devised to study in vitro digestion of proteins. It is a modification of a dialysis cell. The cell consists of an inner reaction vessel fixed into a cylindrical outer compartment where buffer circulation is provided. The vessel is surrounded by a tubular membrane with molecular weight cutoff of 1000. The dimensions of the compartment, the membrane, and the buffer flow rate are determined with labeled amino acids prior to the experiment. For the digestion assay, casein is first hydrolyzed with pepsin at pH 1.9 for 30 minutes. The mixture is then made alkaline (pH 7.5) and poured into the dialysis tube with pancreatin. Nitrogenous material collected with the sodium phosphate buffer will be analyzed directly.
Review of the literature on in vitro approaches for determining bioavailability of contaminants in the human GI tract shows that the two-stage physiologically-based method, the everted sac technique, and the brush border membrane vesicles technique are mostly designed for a single application. They generally include a limited number of simulated parameters, and are not directly applicable for simulating the bioavailability of petroleum (hydrophobic) hydrocarbons in human GI tract. At the same time, review of the literature on in vitro methods from areas of nutrition and pharmacology/pharmacokinetics shows that there are a number of models that will provide a set of tools of varying sophistication. However, because some of these models are designed to be used in medical research, they are too expensive and not practical to be used for testing environmental contamination